When insulin binds to the insulin receptor, this stimulates endocytosis of the hormone-receptor complex. Ligand-stimulated endocytosis requires that the receptor must possess intact tyrosine kinase activity. In this project, we are undertaking to identify other sequence motifs in the intracellular domain of the insulin receptor that are required for endocytosis. Dileucine motifs have been shown to be involved in trans Golgi sorting, lysosomal targeting, and internalization of a number of proteins. Because the insulin receptor contains four dileucine pairs in its cytoplasmic domain, we investigated whether these insulin receptor sequences could serve as lysosomal sorting sequences. Chimeric molecules expressing the Tac antigen fused to each isolated insulin receptor motif were constructed. A chimera containing the juxtamembrane dileucine motif (Glu-Lys-Ile-Thr-Leu-Leu), which closely resembles the sequences originally identified in the gamma- and delta-chains of the T cell receptor, was shown to sort to lysosomes by immunofluorescence microscopy, as did a chimera expressing one dileucine motif (Gly-Gly-Lys-Gly-Leu-Leu) found in the tyrosine kinase domain. Chimeras expressing two other dileucine motifs localized to both lysosomes and the plasma membrane. In contrast, chimeras expressing two other potential sorting signals found in the cytoplasmic tail of the insulin receptor (Asn-Ala-Arg-Asp-Ile-Ile and Lys-Asn-Gly-Arg-Ile-Leu) localized predominantly to the plasma membrane. When alanine residues were substituted for the two leucines in the Glu-Lys-Ile-Thr-Leu-Leu sequence, the resultant chimera was localized exclusively to the cell surface. When the alanine pair was substituted for the two leucine residues in the juxtamembrane domain of the intact insulin receptor and the mutant receptor was expressed in NIH-3T3 cells, the mutation did not impair insulin binding or receptor tyrosine kinase activity. However, the Ala-Ala mutant internalized insulin 80% slower than the wild-type receptor. Taken together, these findings suggest that the dileucine motif found in the juxtamembrane domain of the insulin receptor is involved in receptor internalization. Apparently, other dileucine motifs in the insulin receptor do not completely replace this function of the juxtamembrane dileucine motif. Thus, it is likely that other insulin receptor sequences mask the other potential lysosomal targeting signals in the intact molecule.